Soluble tetramers of major histocompatibility complex (MHC) proteins, charged with specific peptide antigen and fluorescently labeled (“MHC tetramers”), have in the past five years proven extraordinarily useful in the detection, enumeration, characterization and purification of antigen-specific T lymphocytes. Applications of MHC class I tetramers have recently been reviewed in Doherty et al., Annu. Rev. Immunol. 18:561–92 (2000); Ogg et al., Immunol. Lett. 66(1–3):77–80 (1999); Maini et al., Immunol. Today 20(6):262–6 (1999); and Doherty, Curr. Opin. Microbiol. 1(4):419–22 (1998). Applications of MHC class II tetramers are discussed in Reichstetter et al., J. Immunol. 165(12):6994–8 (2000); Kwok et al., J. Immunol. 164(8):4244–9 (2000); Liu et al., Proc. Natl. Acad. Sci. USA 97(26):14596–14601 (2000); Novak et al., J. Clin. Invest. 104(12):R63–7 (1999); Crawford et al., Immunity 8:675–682 (1998); and Kozono et al., Nature 369:151–154 (1994).
MHC tetramers bind to the T cell receptor (“TCR”) of T lymphocytes in an antigen- and MHC-specific manner. See, e.g., Altman et al., Science 274:94 (1996) and U.S. Pat. No. 5,635,363.
Specificity of the staining reagent is conferred by both the MHC moiety and the peptide included within the tetrameric complex. Thus, tetramers comprising the extracellular domains of MHC class I α chain molecules, complexed to β2 microglobulin and charged with antigenic peptides of about 9 amino acids, will bind to class I-restricted, typically CD8+, T cells specific for the charging peptide and MHC allele. Tetramers comprising four class II heterodimers—each heterodimer comprising the extracellular domains of MHC class II α and class II β chains—charged with antigenic peptides, will bind to class II-restricted, typically CD4+, T cells in an antigen-specific and MHC-restricted fashion.
Avidity sufficient to allow stable binding to the T lymphocyte results from the multimerization of the MHC/peptide complex. In order to maintain self-tolerance, the natural affinity of TCR for MHC/peptide is low, too low to permit use of a univalent MHC/peptide complex as an immunological staining reagent. Matsui et al., Science 254:1788–91 (1991); Matsui et al., Proc. Natl. Acad. Sci. USA 91:12862–6 (1994). Multimerization increases the avidity of the MHC/peptide complex for TCR sufficiently to permit stable binding. Typically, multimerization is achieved by enzymatic biotinylation of a BirA substrate peptide engineered into the MHC chain; the biotinylated MHC chain thereafter binds avidin or streptavidin with a 4:1 molar stoichiometry.
The MHC tetramer is rendered detectable by direct or indirect conjugation to a fluorophore. Typically, direct conjugation is accomplished by multimerizing the MHC/peptide molecules using a streptavidin or avidin molecule that has been prior-conjugated to a fluorophore. Such avidin and streptavidin proteins are commercially available from a variety of vendors (e.g., Becton Dickinson Immunocytometry Systems, San Jose, Calif., USA; Biomeda, Foster City, Calif., USA; Ancell Corp., Bayport, Minn. USA; Southern Biotechnology Assocs., Inc., Birmingham, Ala., USA). Indirect labeling can be performed using a fluorophore-conjugated antibody having specificity for the avidin/streptavidin moiety or for nonpolymorphic determinants of the multimerized MHC chain.
Although MHC tetramers have proven tremendously useful, certain aspects of their synthesis present problems.
Proper multimerization requires the controlled, stoichiometric, antecedent biotinylation of the MHC chain, and further requires the subsequent stoichiometric binding of the biotinylated chains to avidin or streptavidin. Inefficiency or imprecision at either or both of these steps affects yield and utility of the resulting tetrameric complex.
For example, inefficient biotinylation of the MHC chain—i.e., failure to incorporate at least one biotin per MHC molecule—will lead to decreased yield of tetramers. Inefficient removal of unconjugated biotin from the reaction products, with carry-over of unconjugated biotin molecules into the multimerization reaction, can saturate avidin molecules and also decrease tetramer yield.
Biotinylation of MHC molecules at multiple sites can reduce the avidity of resulting tetrameric complexes. Early attempts at multimerization using chemical biotinylation failed, possibly because multiple and random chemical modification of MHC molecules led to randomized, and often inactive, orientation of the MHC/peptide moieties in the multimerized complex. Although enzymatic biotinylation has improved control of the biotinylation process, enzymatic modification is itself subject to well known vicissitudes, including dependence upon enzyme concentration, substrate concentration, substrate accessibility, enzyme activity, and the like; multiple enzymatic biotinylation can mimic the poor avidity observed with the early, chemically-biotinylated, complexes.
Biotinylation at multiple sites of the MHC chain, combined with obligate use of an extrinsic multimerizing moiety, such as avidin, also makes possible the spurious generation of higher order multimers during the multimerization reaction. If even a small number of MHC/peptide subunits contain two or more biotins, cross-linking of the avidin/streptavidin moiety can occur. Such higher order multimers can exhibit decreased avidity due to steric hindrance, and can conversely, in other circumstances, lead to higher order complexes having increased avidity. Variations in avidity, whether decreases or increases from the mean, can lead to variations in staining intensity that are unrelated to TCR density or antigen affinity. Higher order multimers can also produce a more intense fluorescent signal, further leading to confounding variations in staining intensity.
Conversely, carry-over of unconjugated biotin molecules into the multimerization reaction can lead to partial saturation of avidin/streptavidin moieties with unconjugated biotin, leading to formation of multimeric complexes having fewer than four MHC/peptide subunits. Such lower order complexes can demonstrate lower avidity for TCR, leading to variations in staining intensity that are unrelated to TCR density or antigen/MHC affinity.
The requirement for a separate fluorophore conjugation step also presents opportunities for yield loss and for unintended variation in either avidity for TCR or fluorescence intensity, or both.
In a related approach, MHC/Ig chimeras, rather than tetramers of MHC, are used to label antigen-specific T lymphocytes. Dal Porto et al., Proc. Natl. Acad. Sci. USA 90:6671–6675 (1993); Greten et al., Proc. Natl. Acad. Sci. USA 95:7568–7573 (1998); Hamad et al., J. Exp. Med. 188:1633–1640 (1998); Schneck et al., U.S. Pat. Nos. 6,015,884 and 6,140,113.
In class I chimeras, the extracellular domains of MHC class I α chain are fused in-frame into the variable region of an IgG heavy chain. Under suitable oxidizing conditions, the heavy chain chimeras self-dimerize through disulfide bonds: dimerization confers avidity for TCR sufficient to permit use of the dimeric fusion protein as a T cell labeling reagent. As with MHC tetramers, the dimeric class I chimera molecule must be associated with β2 microglobulin and charged with specific antigenic peptide to permit recognition of antigen-specific T cells; unlike MHC tetramers, the MHC/Ig chimeric molecules must additionally be associated prior to use with Ig light chains.
In class II MHC/Ig chimeras, the extracellular domains of MHC class II α and β chains are separately fused to Ig heavy and light chain subunits, typically with the β subunit domains fused to IgG heavy chain and the class II α domains fused to Ig light, typically κ, chain. As with MHC tetramers, the class II chimeric molecule must be charged with specific antigenic peptide to permit recognition of antigen-specific T cells; for stability, antigenic peptide can be fused directly to the N terminus of the class II β subunit, Kozono et al., Nature 369:151–154 (1994); Liu et al., Proc. Natl. Acad. Sci. USA 97:14596–14601 (2000).
Labeling of the dimeric chimeras is typically accomplished using a fluorophore-conjugated, anti-Ig, secondary antibody.
The self-dimerizing chimeras obviate the biotinylation step and use of an extrinsic multimerizing moiety required by MHC chimeras, with their attendant problems. The dimeric chimeras also more readily permit post-synthesis loading with antigenic peptide.
However, the dimeric chimeras are not without problems of their own.
First, the dimeric valency can cause lower avidity for TCR than would be obtained using tetramers, reducing or abrogating the ability to label low affinity TCRs.
Second, the additional requirement for Ig light chain association—whether native Ig light chains, as in the class I chimeras, or recombinant Ig light chain fusion proteins, as in the class II chimeras—obligates the coexpression of multiple recombinant expression constructs in a single host cell or, in the case of class I chimeras, use of a host cell line that constitutively expresses Ig light chain.
Third, although self-dimerization obviates the biotinylation step and reliance upon an extrinsic multimerizing moiety of MHC tetramers, potentially improving yield of multimers of known valency (complexity), the dimers can dissociate under reducing conditions.
Additionally, each of the strategies for rendering the dimeric chimera fluorescently detectable presents difficulties.
For example, use of secondary antibodies for visualization can make simultaneous detection of multiple antigens more difficult; this can be a significant problem when, as is often the case, the population of antigen-specific T cells is small, obligating use of multiple parameters in order accurately to detect and enumerate the cells. Direct chemical conjugation of fluorophore to the chimera, an alternative to use of secondary antibodies, can interfere with or even abrogate affinity interactions of the chimera with its cognate TCR. Direct chemical conjugation can also lead to chimeric molecules having varying molar ratios of label, making flow cytometric or other fluorescence-based analyses more problematic.
Thus, there is a need for reagents that can be used to label T lymphocytes based upon their antigen (and MHC) specificity that have sufficient avidity to permit stable binding to T lymphocytes via interactions with surface TCR, but that do not require biotinylation and extrinsic multimerization; there is a concurrent need for antigen-specific T cell labeling reagents that can be rendered fluorescent without requiring use of secondary antibodies or direct chemical conjugation to fluorophore.
The substrate-independent, intrinsically fluorescent green fluorescent protein from Aequorea victoria (“GFP”) has been used in the flow cytometric (“FACS”) analysis of cells that express GFP, or GFP fusions, inside the cell. Reviewed in Galbraith et al., “Flow cytometric analysis and FACS sorting of cells based on GFP accumulation,” Methods Cell Biol. 58:315–41 (1999). GFP variants having characteristics improved for flow cytometry have been described, U.S. Pat. Nos. 6,090,919 and 5,804,387; Cormack et al., Gene 173:33–38 (1996). GFP and GFP variants have typically been used for internal labeling of cells, rather than for external labeling of cell surface structures.
A distant homologue of GFP, termed DsRed (also denominated drFP583), has recently been cloned from Discosoma coral, Matz et al., Nature Biotechnol. 17:969–973 (1999); vectors suitable for excision cloning, bacterial expression and mammalian expression of DsRed, alone or as a fusion protein, are now commercially available (Clontech Laboratories, Inc., Palo Alto, Calif., USA).
Native DsRed has an excitation (absorption) maximum of about 558 nm, and has an emission maximum of about 583 nm, a substantial red shift from the GFP emission maximum of about 509 nm. DsRed is thus readily excited by the standard 488 nm laser line routinely available in flow cytometers and has an emission spectrum readily distinguishable from autofluorescence background and from that of GFP, making DsRed a candidate for two-color flow cytometric applications in conjunction with GFP or FACS-optimized mutants of GFP.
Detailed studies of the structure and spectral properties of DsRed, Baird et al., Proc. Natl. Acad. Sci. USA 97:11984–11989(2000); Gross et al., Proc. Natl. Acad. Sci. USA 97:11990–11995 (2000); Heikal et al., Proc. Natl. Acad. Sci. USA 97:11996–12001 (2000); Wall et al., Nature Structural Biol. 7:1133–1138 (2000), however, have identified two characteristics that are said to militate against widespread use of DsRed in flow cytometric and other fluorescence analyses.
First, the red fluorescence of DsRed is slow to mature, requiring days to ripen fully from green to red, both in vitro and in vivo. A maturation time on the order of days will preclude attempts to use DsRed as a reporter of short-term gene expression or to track fusion proteins in organisms with short generation times or fast development. Baird et al., Proc. Natl. Acad. Sci. USA 97:11984–11989 (2000).
Second, DsRed appears to be an obligate, and self-multimerizing, tetramer. Baird et al., Proc. Natl. Acad. Sci. USA 97:11984–11989 (2000); Wall et al., Nature Structural Biol. 7:1133–1138 (2000); Gross et al., Proc. Natl. Acad. Sci. USA 97:11990–11995 (2000). Although oligomerization may be irrelevant to use of DsRed as a simple reporter of gene expression, it will present serious problems in most potential applications where DsRed would be fused to a host protein to report on the trafficking or interactions of the latter. Furthermore, many proteins in signal transduction are activated by oligomerization; fusion to DsRed could cause constitutive signaling. For host proteins that are already oligomeric, fusion to DsRed could either cause clashes of stoichiometry, steric conflicts of quaternary structures, or cross-linking into massive aggregates.
Given these “major drawbacks,” “[m]any potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.” Baird et al., Proc. Natl. Acad. Sci. USA 97:11984–11989 (2000). “For most biotechnological applications, both the slow maturation and oligomerization of DsRed are undesirable properties that must be addressed through systematic mutagenesis.” Wall et al., Nature Structural Biol. 7:1133–1138 (2000). “The excellent brightness and stability of DsRed and many potential uses for a long-wavelength fluorescent protein provide ample justification for major efforts to remedy these remaining deficiencies.” Gross et al., Proc. Natl. Acad. Sci. USA 97:11990–11995 (2000).